Neuronal cell death and axonal damage are hallmarks of many neurological disorders and neurodegenerative diseases including, Alzheimer's and Parkinson's diseases, spinal cord injury, glaucoma, multiple sclerosis, traumatic and ischemic brain injuries, and over 100 different peripheral neuropathies. The dual leucine zipper kinase (DLK, MAP3K12) is a key sensor of axon injury and contributes to three distinct neuronal outcomes: (1) cell death, (2) axon degeneration (AxD), and (3) axon regeneration (AxR). Thus DLK signaling is an exciting new therapeutic target for a wide range of neurological disorders and injury. In spite of the clinical importance for targeting DLK signaling, we know shockingly little about how DLK is regulated and contributes to neuronal outcome after injury. This proposal will address this fundamental gap in our knowledge and will identify regulators of DLK and test their roles in neuronal injury signaling and outcomes. I performed a pilot screen to identify proteins that restrict DLK signaling in DRG neurons, and identified MAP3K12 binding inhibitor protein (MBIP) as a negative regulator of DLK. In Aim 1, I will determine the mechanism by which MBIP regulates DLK signaling. This will involve genetic and pharmacological manipulation of DLK signaling and assessment of each level of the pathway. In Aim 1, I will also test the hypothesis that injury relieves MBIP-inhibition of DLK and manipulating MBIP is sufficient to alter the injury phenotype in models of DLK-dependent cell death, AxD, and AxR. In Aim 2, I will test the hypothesis that MBIP regulates basal DLK-signaling in vivo by assessing spontaneous DLK pathway activation. Furthermore, I will test the role of MBIP in peripheral nerve injury. This will be accomplished using multiple readouts of AxD and AxR in the sciatic nerve and re-innervation of hindlimb muscles. Aims 1 and 2 will (1) elucidate the mechanism by which MBIP regulates DLK signaling and how this is affected by injury, (2) test the therapeutic potential of targeting MBIP after injury, and (3) determine the role of MBIP-mediated inhibition of DLK signaling in vivo. Due to the success of my pilot screen, I propose a larger candidate-based screen to identify additional positive and negative regulators of DLK in Aim 3. Candidates will be evaluated for their ability to suppress or enhance molecular markers of the DLK signaling pathway. Hits will be further evaluated by testing their contributions to neuronal outcome in multiple injury models. This aim will identify novel regulators of the DLK pathway in mammalian sensory neurons and will lead to testable hypotheses to uncover mechanisms of DLK regulation and the impact on injury phenotypes. These aims will fill a critical gap in knowledge regarding mechanisms of DLK regulation and the relationship between DLK regulators and neuronal injury phenotypes. These results will significantly enhance our understanding of DLK signaling and may identify novel molecular targets for therapeutic intervention in a wide range of neuronal injuries and diseases.